This invention relates generally to proteomics, and more specifically to detection of protease activity in formats amenable to multiplex analysis.
Proteases are a class of enzymes with important roles in the regulation of cellular activity. Proteases act by cleaving proteins into smaller versions. This results in activation or inactivation of the proteins which in turn influences the role of the proteins in diverse physiological processes. Examples of physiological processes that are directly affected by proteases include blood coagulation, inflammation, programmed cell death, reproduction, fibrinolysis, and immune response. Numerous disease states are caused by, or can be characterized by, the alterations in the activity of specific proteases. The importance and complexity of the roles played by proteases in human health is evident in the fact that there are at least 575 known and putative proteases in humans, and it is estimated that up to 1,200 human genes encode proteases. The ability to detect and evaluate these proteases in research or clinically is significant to the investigation, treatment, and management of disease states.
In addition, there are many pathogenic microorganisms that depend upon specific protease activity for their infectivity. As an example, many viruses depend on protease cleavage of inactive precursors, called pro-proteins, to form active products that mediate infection. Inhibiting such selective cleavage can inhibit the viability of the virus. Again, the ability to evaluate these proteases in a research or clinical setting would benefit investigation, treatment and management of many pathogenic diseases.
Several blockbuster drugs that have been introduced to the market are protease inhibitors. For example, protease inhibitor drugs have been developed to inhibit viral proteases required for replication of HIV and in many cases have been the most effective treatments for HIV/AIDS. Other protease inhibitor drugs block the human protease, thrombin, which is involved in blood clotting and are among the most effective treatments for stroke and coronary infarction. Protease inhibitor drugs are also used to treat high blood pressure. Overall, it has been estimated that 5-10% of all pharmaceutical targets are proteases. In this regard, other protease inhibitors are being developed to treat parasitic, fungal, and viral infections; inflammatory, immunological, and respiratory conditions; cardiovascular and neurodegenerative disorders including Alzheimer's disease; and cancers.
Proteases are also important in food processing. Some well known examples are the production of cheese which has traditionally relied upon proteases isolated from the stomach of unweaned calves and meat tenderization which traditionally utilized papain from the leaves and unripe fruit of Carica papaya. Proteases are also used in the baking industry. For example, pastry dough may be prepared more quickly if its gluten is partially hydrolyzed using a heat-labile protease that is inactivated early in the subsequent baking. Although several traditional methods are still practiced in food processing, there is a need for new proteases that provide more efficient food production or new varieties and flavors. For example, the amount and types of proteases used during cheese ripening has substantial effects on flavor and quality. As such differences in proteases used for cheese ripening is largely responsible for the distinct varieties of cheeses available.
In a further example, proteolysis of inexpensive materials such as soya protein can increase the range and value of their usage in producing new foods. In this regard, partial hydrolysis of soya protein can greatly increase its ‘whipping expansion’ whereas further hydrolysis can improve its emulsifying capacity. Proteases can also be used to recover protein from parts of animals that would otherwise go to waste after butchering. For example, residual meat on manually butchered bones can be removed by proteases and the resulting meat slurry used to provide canned meats and soups.
Traditionally, however proteases have not always been easy to evaluate and identify. Although specific assays have been developed to measure activity of individual proteases, their utility for evaluating proteases in more complex biological mixtures has been limited. Furthermore, the ability to identify new substrates or inhibitors for proteases is often difficult using assays for individual proteases. The result is inefficient identification of useful proteases and or inhibitors.
Thus, there exists a need for assays that allow the rapid and efficient analysis of protease activity in complex biological mixtures. There is also a need for assays to identify previously unknown protease substrates or inhibitors. The present invention satisfies these needs and provides other advantages as well.